CN101887060A - Kit for quantitatively detecting concentration of phenylalanine in blood of neonate by using fluorescence developing - Google Patents
Kit for quantitatively detecting concentration of phenylalanine in blood of neonate by using fluorescence developing Download PDFInfo
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Abstract
The invention provides a kit (fluorescence method) for quantitatively detecting phenylalanine, and belongs to the technical fields of biology, medicaments and medical equipment. The kit is characterized in that: 1, the quantitative detecting kit for screening neonatal phenylketonuria overcomes the defects of semiquantitative determination, hard automation and interference by antibiotics of the bacterial inhibition assay; and 2, the kit replaces aseptic filtration by a 0.22mu m microfiltration membrane to avoid harm to a user body and environment and avoid potential risk of reagents in the transporting process, and replaces the extraction experiment by using high-concentration alcohol in conventional imported reagents, wherein the high-concentration alcohol has high volatility and causes a big error to the experimental result. Therefore, the big error to the experimental result caused by the reagents per se can be avoided and the using risk can be reduced by using combination of trichloroacetic acid with double distilled water instead of the high-concentration alcohol extraction method.
Description
Technical field
The present invention relates to clinical diagnosis reagent application apparatus technical field, belong to a kind of kit that utilizes concentration of phenylalanine in the quantitative special detection blood of neonate of fluorescence developing
Background technology
At present, known now domestic be used for detecting the blood of neonate concentration of phenylalanine the most widely experimental technique be that bacterium suppresses method and fluorescence method.
The deficiency that bacterium inhibition method exists, this method is a kind of quantivative approach of doing, and subjectivity is strong, and sensitivity is low, experimental period is long etc., in addition, and the artificial treatment check data, manual registration inspection record is unfavorable for the storage of data and for future reference, brings potential risk for the standardized management of newborn child's illness center.
The present domestic fluorescence developing that adopts detects neonate's detection kit all by external imported product, often with the supporting use of particular detection instrument, and can not be used for other detecting instrument, and this kit and institute's necessary instrument equipment price costliness, be unfavorable for popularizing of neonatal screening, non-general examination of newborn infant diseases center can be born.In addition, the required extract of import reagent box is that the alcohol volatilization of high concentration is very strong, and the experimental result error is very big, and the import reagent box is added with domestic taboo control products Sodium azide as antiseptic in liquid dosage.In practical operation, the people had certain danger.
Summary of the invention
The purpose of this invention is to provide a kind of result accurately and reliably, non-invasive, safe, highly sensitive, neonatal screening kit that use cost is low.This kit is a kind of fluorescent quantitation based on 96 hole micro-reaction plates, can on any fluorescence detector device, use, and improved the concentration of extract, and make experimental result more accurate, the cost of this kit is 1/4th of an import reagent box in addition.Can promote the extensive examination of neonate's phenylketonuria.Each neonate can both be checked.Reduce the pressure of country and society, make our contribution for improving whole people's population quality.
For achieving the above object, technical solution of the present invention is: a kind of kit that utilizes concentration of phenylalanine in the fluorescence developing detection by quantitative blood of neonate is characterized in that: described kit is made up of succinic acid damping fluid, two peptide solutions, ninhydrin solution, copper solution, extract, standard blood sheet, Quality Control blood sheet, 96 hole microwell plates, epiphragma.
The compound method of above-mentioned succinic acid damping fluid is:
Accurately take by weighing the succinic acid aequum with analytical balance, add volumetric flask, the amount that adds distilled water 80% is dissolved, transfer pH value to 5.8 ± 0.01, carry out constant volume with distilled water, with 0.22um specification miillpore filter the succinic acid damping fluid is filtered, amount is divided and to be installed in the reagent bottle according to the rules again, tightens bottle cap and puts into 2～8 ℃ of refrigerators and preserve standby.
This reagent is with external kit difference, we development personnel adopt 0.22um specification miillpore filter in 100,000 grades of operation rooms the succinic acid damping fluid to be carried out aseptic filtration in aseptic this link of this damping fluid of maintenance, have substituted the method for in the past using the Sodium azide antiseptic.Purpose is: 1) to the preservation of this reagent long period; 2) avoid owing to use the Sodium azide antiseptic to cause the harm of human body, environment; 3) avoided reagent in the dangerous hidden danger of transportation.
Above-mentioned two peptide solutions comprise L-leucine, L-alanine, and its compound method is:
(1) accurately takes by weighing L-leucine, L-alanine aequum with analytical balance, add volumetric flask;
(2) amount of adding distilled water 80% is dissolved.After treating to dissolve fully, carry out constant volume with distilled water at last, obtain two peptide solutions.Two peptide solutions that prepare are measured branch according to the rules install in the reagent bottle, tighten bottle cap and put into 2～8 ℃ of refrigerators and preserve standby.
Described ninhydrin solution preparation method:
With analytical balance weighing triketohydrindene hydrate,, after the dissolving, get final product fully with the distilled water dissolving.
Described copper solution compound method:
With the accurate weighing sodium carbonate of analytical balance, be dissolved in distilled water (80% amount), tighten bottle cap and put into 2～8 ℃ of refrigerators and preserve standby
Accurately take by weighing tartrate Ghana with analytical balance and be dissolved in distilled water (80% amount), tighten bottle cap and put into 2～8 ℃ of refrigerators and preserve standby.
Accurately take by weighing copper sulphate with analytical balance and be dissolved in distilled water (80% amount), tighten bottle cap and put into 2～8 ℃ of refrigerators and preserve standby.
Behind three solution that prepare respectively, by being that copper sulphate is poured natrium carbonicum calcinatum in proper order, winestone
In acid potassium sodium two kinds of mixed liquors, magnetic stirring apparatus does not stop to stir, and avoids producing precipitation, mixes back distilled water constant volume and get final product, and amount is divided and installed in the reagent bottle according to the rules, and tightening bottle cap, to put into 2～8 ℃ of refrigerators preservations standby.
Described extract compound method:
Accurately take by weighing 30% trichloroacetic acid solid of required preparation total amount with analytical balance, put into an amount of beaker, the amount that adds distilled water 70% is fully dissolved.Carry out constant volume, the trichloroacetic acid solution for preparing is measured branch according to the rules install in the reagent bottle, tighten bottle cap and put into 2～8 ℃ of refrigerators and preserve standby.
Be that with external kit difference the required extract of import reagent box is the alcohol of high concentration during this reagent preparation, it is very strong to volatilize, and the experimental result error is very big, causes certain risk to the user.And our performers and clerks to use be that trichloroacetic acid and distilled water are in conjunction with making extract.Purpose is: 1) avoided because the resultant error that the technical reason of reagent own causes is bigger than normal.Reduce user's risk.2) this reagent can be preserved down with other matched reagent similarity condition, reduces production costs.
Described standard, Quality Control blood piece preparation method:
With whole blood 4-8 layer filtered through gauze, centrifugal then 3000 changeed 20 minutes, abandoned or adopted blood plasma, washed 3 times with physiological saline again, and 3000 left the heart 20 minutes, abandoned or adopted supernatant, and last centrifugal 4000 changeed 30 minutes, mixed up ratio with the 60g/L bovine serum albumin(BSA), mixing
Surveying hematocrit is that 55-65% is advisable
Blood cell by A-F standard and Quality Control C1-C2 standard, the quality-control product for preparing, is added in the blood cell in the desired amount, and mixing can titrimetric standard Quality Control blood sheet.
Described using method may further comprise the steps:
Phenylalanine is measured:
1. before the experiment kit is placed on room temperature (18～25 ℃) balance earlier, copper solution still is placed in 2～8 ℃ of refrigerators, promptly gets promptly and uses.、
2. reaction mixture: succinic acid damping fluid, two peptide solutions and indenes three copper solutions are mixed in 5: 1: 2 ratios, keep in Dark Place, and use up in 4 hours.
3. lay the filter paper blood cake of diameter 3mm standard items, quality-control product, sample with special-purpose punching device, put into the corresponding hole of transparent microwell plate;
4. every hole adds the 80ul extract, epiphragma, room temperature vibration 30 minutes (avoiding blood sheet bob during vibration, in order to avoid extraction not exclusively);
5. every hole is taken out extract 50ul and is transferred in the corresponding hole of white microwell plate;
6. every hole adds reaction mixture 50ul, and epiphragma vibrated 15 seconds, 60 ℃ of incubations 1 hour or 37 ℃ of incubations 2 hours;
7. from the incubation case, take out microwell plate, put into refrigerator (18～-20 ℃) freezing 3～5 minutes;
8. the copper solution 200ul of every Kong Jialeng (2～8 ℃ refrigerators in take out), lucifuge was room temperature incubation 15～30 minutes;
9. the machine of going up is measured, the excitation wavelength 390nm that luminoscope uses, emission wavelength 486nm.
The explanation of phenylalanine measurement result
In the mixed liquor that contains succinic acid damping fluid, two peptide solutions and ninhydrin solution, triketohydrindene hydrate can react with phenylalanine, produce a kind of fluorescent material, wherein dipeptides can improve this fluorescence reaction, succinic acid damping fluid control PH 5.8 ± 0.1, desirable fluorescent value and maximum specificity can be guaranteed, the stable more and cessation reaction of fluorescent chemicals can be made after the adding copper solution.Use fluorescence readout instrument (exciting light 390nm, emission light 486nm) to measure the power of fluorescent material, can reflect the concentration of phenylalanine.In the mixed liquor that contains succinic acid damping fluid, two peptide solutions and ninhydrin solution, triketohydrindene hydrate can react with phenylalanine, produce a kind of fluorescent material, wherein dipeptides can improve this fluorescence reaction, succinic acid damping fluid control PH 5.8 ± 0.1, desirable fluorescent value and maximum specificity can be guaranteed, the stable more and cessation reaction of fluorescent chemicals can be made after the adding copper solution.Adding can reduce experimental error with the extract that trichloroacetic acid makes, use fluorescence readout instrument (exciting light 390nm, emission light 486nm) power of mensuration fluorescent material, the concentration of detection phenylalanine adopts data analyzing and processing software that data are handled then.Realize the purpose of neonate's examination in 72 hours, owing to used chemiluminescent quantitative measurement technology, the result has realized non-invasive, safe, highly sensitive neonatal screening accurately and reliably, and the present invention has higher market popularization value.
Points for attention:
1, the reagent of different lot numbers can not be used with;
2, this product is a disposable product;
3, the reagent that exceeds the term of validity can not be used again;
4, all reagent and samples will reach room temperature (18～26 ℃) before use;
5, (from 2～8 ℃ of refrigerators, promptly get promptly and use) except the copper solution;
6, when from standard items, when quality-control product punches, note from the low concentration to the high concentration.
7, experiment finishes all article, adopts immersion treatment (biological products should be considered as potential hazard) such as high temperature, Peracetic acid or 84 liquid.
8, the concentration of standard, quality-control product will send advice note to tell by telephone simultaneously with kit as changing in the kit.
The Figure of description explanation:
1, Fig. 1: whole kit outside drawing;
2, Fig. 2: kit vertical view;
3, Fig. 3-Fig. 7: reagent solution carrying bottle;
4, Fig. 8-Fig. 9: detect and use microwell plate;
5, Figure 10: standard blood sheet.
Claims (7)
1. the external diagnosis reagent case of neonate's concentration of phenylalanine in the detection by quantitative filter paper dried blood spot, it is characterized in that: described reagent is made up of succinic acid damping fluid, two peptide solutions, ninhydrin solution, copper solution, extract, 96 hole microwell plates (transparent), standard blood sheet.
2. the preparation method of phenylalanine detection by quantitative kit as claimed in claim 1 is characterized in that: described succinic acid damping fluid compound method:
(1) accurately takes by weighing the succinic acid aequum with analytical balance, put into an amount of beaker;
(2) 80% of adding preparation total amount distilled water dissolves;
(3) transfer pH value to 5.8 ± 0.01, carry out constant volume with distilled water;
(4) with 0.22um specification miillpore filter the succinic acid damping fluid is filtered again, measure branchs according to the rules and install in the reagent bottle filtering good succinic acid damping fluid, tightening bottle cap, to put into 2～8 ℃ of refrigerators preservations standby.
Be with external kit difference during the preparation of succinic acid damping fluid described in this right, we development personnel adopt 0.22um specification miillpore filter in 100,000 grades of operation rooms the succinic acid damping fluid to be filtered in aseptic this link of this damping fluid of maintenance, have replaced the Sodium azide preserving method that in the past used.Purpose is: 1) to the preservation of this reagent long period.2) avoid causing the harm of human body, environment.3) avoided reagent in the dangerous hidden danger of transportation.
3. as the preparation method of phenylalanine detection by quantitative kit as described in the claim 2, it is characterized in that: described two peptide solutions comprise L-leucine, L-alanine, its compound method:
(1) accurately takes by weighing L-leucine, L-alanine aequum with analytical balance, add volumetric flask;
(2) amount of the distilled water 80% of adding preparation total amount is dissolved.After treating to dissolve fully, carry out constant volume with distilled water at last, obtain two peptide solutions.Two peptide solutions that prepare are measured branch according to the rules install in the reagent bottle, tighten bottle cap and put into 2～8 ℃ of refrigerators and preserve standby.
4. as the preparation method of phenylalanine detection by quantitative kit as described in the claim 3, it is characterized in that: described ninhydrin solution, its compound method is:
(1) with the accurate weighing triketohydrindene hydrate of analytical balance aequum;
(2) amount of the distilled water 80% of adding preparation total amount is dissolved, and after treating to dissolve fully, carries out constant volume with distilled water at last, obtains ninhydrin solution.Indenes three solution that prepare are measured branch according to the rules to install to and tightens bottle cap in the reagent bottle and put into 2～8 ℃ of refrigerators and preserve standby.
5. as the preparation method of phenylalanine detection by quantitative kit as described in the claim 4, it is characterized in that: described copper solution comprises sodium carbonate liquor, tartrate Ghana solution and copper-bath, and its compound method is:
(1) preparation of sodium carbonate liquor:
With the accurate weighing sodium carbonate aequum of analytical balance, add volumetric flask, add 80% amount distilled water, fully after the dissolving, put into 2～8 ℃ of refrigerators and preserve standby the solution for preparing is packaged.
(2) preparation of tartrate Ghana solution:
Accurately take by weighing tartrate Ghana aequum with analytical balance, add volumetric flask, add 80% amount distilled water, fully after the dissolving, put into 2～8 ℃ of refrigerators and preserve standby the solution for preparing is packaged;
(3) preparation of copper-bath:
Accurately take by weighing the copper sulphate aequum with analytical balance, add 80% amount distilled water, fully after the dissolving, put into 2～8 ℃ of refrigerators and preserve standby the solution for preparing is packaged;
(4) preparation of copper solution:
With three stock solution preparing by along being that copper sulphate is poured natrium carbonicum calcinatum in proper order, in two kinds of mixed liquors of sodium potassium tartrate tetrahydrate, magnetic stirring apparatus does not stop to stir, fully after the dissolving, use the distilled water constant volume, the solution for preparing is measured branch according to the rules install in the reagent bottle, tighten bottle cap and be placed on 2～8 ℃ of refrigerators and preserve standby.
6. as the preparation method of phenylalanine detection by quantitative kit as described in the claim 5, it is characterized in that: described extract compound method is:
1) with 30% trichloroacetic acid solid of the accurate weighing of analytical balance preparation total amount, puts into an amount of beaker;
2) after an amount of distilled water dissolving of adding, carry out constant volume;
3) extract for preparing is measured branch according to the rules and install in the reagent bottle, stubborn dead bottle cap is placed on 2～8 ℃ of refrigerators and preserves standby.
Be with external kit difference during the preparation of extract described in this right that the required extract of import reagent box is that the alcohol volatilization of high concentration is very strong, the experimental result error is very big.And our development personnel use is that trichloroacetic acid and distilled water are in conjunction with making extract.Purpose is: 1) avoided the resultant error that causes owing to the reason of reagent own bigger than normal.2) can preserve equally with other matched reagent, reduce production costs.
7. as the preparation method of phenylalanine detection by quantitative kit as described in the claim 6, it is characterized in that: described standard blood sheet, Quality Control blood sheet the preparation method be:
(1) whole blood is used 4-8 layer filtered through gauze;
(2) centrifugal then 3000 changeed 20 minutes, abandoned or adopted blood plasma;
(3) wash 3 times with physiological saline, 3000 left the heart 20 minutes, abandoned or adopted supernatant again;
(4) last centrifugal 4000 changeed 30 minutes, mixed up ratio with the 60g/L bovine serum albumin(BSA), and it is 55-65% that mixing is surveyed hematocrit;
(5) blood cell being divided into 8 parts of phenylalanine (A-F) standards that add variable concentrations drips on the 903# filter paper according to different concentration with the standard for preparing with Quality Control (C1-C2), naturally dry in the shade (2-4 hour), put in the polybag that drying agent is housed the 2-8 degree and store standby.
Phenylalanine is measured:
1. before the experiment kit is placed on room temperature (18 ℃～25 ℃) balance earlier, copper solution still is placed in 2～8 ℃ of refrigerators, promptly gets promptly and uses.
2. reaction mixture: succinic acid damping fluid, two peptide solutions and indenes three copper solutions are mixed in 5: 1: 2 ratios, keep in Dark Place, and use up in 4 hours;
3. lay the filter paper blood cake of diameter 3mm standard items, quality-control product, sample with special-purpose punching device, put into the corresponding hole of transparent microwell plate;
4. every hole adds the 80ul extract, epiphragma, room temperature vibration 30 minutes (avoiding blood sheet bob during vibration, in order to avoid extraction not exclusively);
5. every hole is taken out extract 50ul and is transferred in the corresponding hole of white microwell plate;
6. every hole adds reaction mixture 50ul, and epiphragma vibrated 15 seconds, 60 ℃ of incubations 1 hour or 37 ℃ of incubations 2 hours;
7. from the incubation case, take out microwell plate, put into refrigerator (18～-20 ℃) freezing 3～5 minutes;
8. the copper solution 200ul of every Kong Jialeng (2～8 ℃ refrigerators in take out), lucifuge was room temperature incubation 15～30 minutes;
9. the machine of going up is measured, the excitation wavelength 390nm that luminoscope uses, emission wavelength 486nm.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101366866A CN101887060A (en) | 2009-05-13 | 2009-05-13 | Kit for quantitatively detecting concentration of phenylalanine in blood of neonate by using fluorescence developing |
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| CN2009101366866A CN101887060A (en) | 2009-05-13 | 2009-05-13 | Kit for quantitatively detecting concentration of phenylalanine in blood of neonate by using fluorescence developing |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102323259A (en) * | 2011-06-19 | 2012-01-18 | 浙江大学 | Method for batch-determining content of amino acid by using chimney-top 96-well PCR (Polymerase Chain Reaction) plate |
| CN102608329A (en) * | 2012-03-08 | 2012-07-25 | 郭健 | Maternal amniotic fluid phenylalanine detection method for phenylketonuria (PKU) antenatal diagnosis |
| CN102798691A (en) * | 2011-05-21 | 2012-11-28 | 中国科学院大连化学物理研究所 | Novel method for detecting phenylalanine dipeptide in blood |
| CN103196897A (en) * | 2013-02-22 | 2013-07-10 | 北京源德生物医学工程有限公司 | Quantitative detection kit and quantitative detection method for phenylalanine |
| WO2014110887A1 (en) * | 2013-01-21 | 2014-07-24 | 广州市丰华生物工程有限公司 | Blood slide bracket, blood slide bracket assembly, filter paper dried blood slide punching machine, and application of blood slide bracket in in-vitro diagnostic reagent |
| CN105445468A (en) * | 2014-08-19 | 2016-03-30 | 苏州新波生物技术有限公司 | Phenylalanine detection kit |
| CN110441524A (en) * | 2019-07-22 | 2019-11-12 | 浙江大学 | Detect application of the antibody of phenylketonuria marker in preparation diagnosis test paper |
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2009
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102798691A (en) * | 2011-05-21 | 2012-11-28 | 中国科学院大连化学物理研究所 | Novel method for detecting phenylalanine dipeptide in blood |
| CN102323259A (en) * | 2011-06-19 | 2012-01-18 | 浙江大学 | Method for batch-determining content of amino acid by using chimney-top 96-well PCR (Polymerase Chain Reaction) plate |
| CN102608329A (en) * | 2012-03-08 | 2012-07-25 | 郭健 | Maternal amniotic fluid phenylalanine detection method for phenylketonuria (PKU) antenatal diagnosis |
| WO2014110887A1 (en) * | 2013-01-21 | 2014-07-24 | 广州市丰华生物工程有限公司 | Blood slide bracket, blood slide bracket assembly, filter paper dried blood slide punching machine, and application of blood slide bracket in in-vitro diagnostic reagent |
| CN103196897A (en) * | 2013-02-22 | 2013-07-10 | 北京源德生物医学工程有限公司 | Quantitative detection kit and quantitative detection method for phenylalanine |
| CN105445468A (en) * | 2014-08-19 | 2016-03-30 | 苏州新波生物技术有限公司 | Phenylalanine detection kit |
| CN110441524A (en) * | 2019-07-22 | 2019-11-12 | 浙江大学 | Detect application of the antibody of phenylketonuria marker in preparation diagnosis test paper |
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Application publication date: 20101117 |